Cyanovirin-N (CV-N) is a potent HIV-inactivating protein that was originally isolated and identified from aqueous extracts of the cultured cyanobacterium Nostoc ellipsosporum (U.S. Pat. No. 6,420,336). Since its identification, methods have been developed for the recombinant production of cyanovirin-N in Escherichia coli (Mori, T. et al., Protein Expr. Purif. 12:151-158, 1998). Cyanovirin-N is an 11 kDa protein consisting of a single 101-amino acid chain containing two intra-chain disulfide bonds. CV-N is an elongated, largely β-sheet protein that displays internal two fold pseudosymmetry and binds with high affinity and specificity to the HIV surface envelope protein, gp120 (Bewley, C. R. et al, Nature Structural Biology 5(7):571-578, 1998).
Despite its observed anti-viral activity, development of cyanovirin-N protein therapies has been hampered by its relatively short half-life after administration, as well as its in-vivo immunogenicity and potential toxic side effects. Most proteins, particularly relatively low molecular weight proteins introduced into the circulation, are cleared quickly from the mammalian subject by the kidneys. This problem may be partially overcome by administering large amounts of a therapeutic protein or through frequent dosing. However, higher doses of a protein can elicit antibodies that can bind and inactivate the protein and/or facilitate the clearance of the protein from the subject's body. In this way, repeated administration of such therapeutic proteins can essentially become ineffective. Additionally, such an approach may be dangerous since it can elicit an allergic response.
Various attempts to solve the problems associated with protein therapies include microencapsulation, liposome delivery systems, administration of fusion proteins, and chemical modification. The most promising of these to date is modification of a therapeutic protein by covalent attachment of poly(alkylene oxide) polymers, particularly polyethylene glycols (“PEG”). For example, Roberts, M. et al., Adv. Drug Delivery Reviews 54 (2002), 459-476, describes the covalent modification of biological macromolecules with PEG to provide physiologically active, non-immunogenic water-soluble PEG conjugates. Methods of attaching PEG to therapeutic molecules, including proteins, are also disclosed in, for example, U.S. Pat. Nos. 4,179,337, 5,122,614, 5,446,090, 5,990,237, 6,214,966, 6,376,604, 6,413,507, 6,495,659, and 6,602,498, each of which is incorporated herein by reference.
The hydrated random coil nature of PEG masks surface epitopes on proteins that would otherwise be recognized by the immune system. As a result, attachment of PEG to a therapeutic protein can slow its rejection by the body, reduce protein, cell and bacterial adsorption, and increase the hydrodynamic radius of the protein to reduce glomerular filtration and kidney clearance. Several proteins have been modified by addition of PEG, including adenosine deamidase, L-asparaginase, interferon alpha 2b, superoxide dismutase, streptokinase, tissue plasminogen activator (tPA), urokinase, uricase, hemoglobin, interleukins, interferons, TGF-β, EGF, and other growth factors, to name a few (Nucci et al, Adv. Drug Delivery Rev. 4:133-151,1991). Such modification has provided extended half-lives of the proteins, reduced toxicity and/or immunogenicity, improved pharmacokinetics, and greater solubility compared to the unconjugated proteins.
Unfortunately, attachment of polymer chains such as PEG to a protein does not, in all cases, result in a protein having improved therapeutic properties. During PEGylation, if the modification of the protein goes substantially to completion, i.e. if all or a majority of the available reactive sites on the protein are PEGylated, a significant amount of the bioactivity of the protein can be lost. For example, as described below, PEGylation of the lysine residues of cyanovirin-N produced conjugates having no significant bioactivity.
Partial PEGylation of a protein can reduce this impact on bioactivity. However, a drawback of partial modification, when using a non-selective process, is the production of a heterogeneous mixture of PEGylated protein, having a statistical distribution of various PEGylated species, e.g., mixtures of mono-PEGylated, di-PEGylated species and the like, at various available residue positions within the protein. It is difficult to predict with any certainty the impact of such attachment upon the properties of the resulting conjugate composition (e.g., stability, bioactivity, toxicity, etc.).
Moreover, such randomly PEGylated conjugate compositions, containing a mixture of PEGylated proteins differing in both the number and position of the PEG groups attached, often cannot be reproducibly prepared. Such mixtures of diversely modified proteins are generally not suitable for use as pharmaceutical compositions.
Purification and isolation of a defined class of PEGylated proteins from such a mixture, even when feasible, involves time-consuming and expensive procedures which result in an overall reduction in the yield of the specific PEGylated protein of interest. Separation of positional isomers, i.e. conjugates containing the same number of PEG moieties but at different positions, can be especially difficult, since they have similar molecular weights. These complications can render use of non-specifically PEGylated proteins economically impractical.
Due to the above described drawbacks to many of the existing PEGylation approaches, there remains a need to develop approaches for attaching PEG to specific molecules, such as cyanovirin, to provide PEG conjugates that significantly retain their bioactivity while exhibiting reduced systemic toxicity and improved circulating half-life, and result in pharmaceutical compositions having well-defined components.